Transcriptional responses of cultured rat sympathetic neurons during BMP-7-induced dendritic growth

Abstract

Background: Dendrites are the primary site of synapse formation in the vertebrate nervous system; however, relatively little is known about the molecular mechanisms that regulate the initial formation of primary dendrites. Embryonic rat sympathetic neurons cultured under defined conditions extend a single functional axon, but fail to form dendrites. Addition of bone morphogenetic proteins (BMPs) triggers these neurons to extend multiple dendrites without altering axonal growth or cell survival. We used this culture system to examine differential gene expression patterns in naïve vs. BMP-treated sympathetic neurons in order to identify candidate genes involved in regulation of primary dendritogenesis.

Methodology/principal findings: To determine the critical transcriptional window during BMP-induced dendritic growth, morphometric analysis of microtubule-associated protein (MAP-2)-immunopositive processes was used to quantify dendritic growth in cultures exposed to the transcription inhibitor actinomycin-D added at varying times after addition of BMP-7. BMP-7-induced dendritic growth was blocked when transcription was inhibited within the first 24 hr after adding exogenous BMP-7. Thus, total RNA was isolated from sympathetic neurons exposed to three different experimental conditions: (1) no BMP-7 treatment; (2) treatment with BMP-7 for 6 hr; and (3) treatment with BMP-7 for 24 hr. Affymetrix oligonucleotide microarrays were used to identify differential gene expression under these three culture conditions. BMP-7 significantly regulated 56 unique genes at 6 hr and 185 unique genes at 24 hr. Bioinformatic analyses implicate both established and novel genes and signaling pathways in primary dendritogenesis.

Conclusions/significance: This study provides a unique dataset that will be useful in generating testable hypotheses regarding transcriptional control of the initial stages of dendritic growth. Since BMPs selectively promote dendritic growth in central neurons as well, these findings may be generally applicable to dendritic growth in other neuronal cell types.

Figure 1. BMP-7-induced dendritic growth in cultured sympathetic neurons requires transcription.

Sympathetic neurons dissociated from embryonic rat superior cervical ganglia (SCG) and cultured in defined medium in the absence of serum and non-neuronal cells were treated with BMP-7 (50 ng/ml) added to the medium on day 5 in vitro. (A) Cultures were fixed at varying times after BMP-7 addition and immunostained for MAP-2 to visualize dendritic processes. Significant dendritogenesis was evident within 48 hr after BMP-7 addition, and by 96 hr, over 95% of the neurons had responded to the dendrite-promoting activity of BMP-7. (B) Actinomycin-D (100 ng/ml) was added to sympathetic cultures at varying times after addition of BMP-7 (50 ng/ml). Following a 72 hr exposure to BMPs, dendritic growth was quantified in MAP-2 immunoreactive cells. Actinomycin-D inhibited dendritogenesis when added within 24 hr after BMP-7 treatment. BMP-7-induced dendritic growth was not blocked when actinomycin-D was added at 48 or 72 hr after BMP-7 treatment. Data presented as the mean ± S.E. (n = 3). *Significantly different from control (cultures grown in the absence of BMP-7) at p<0.001 (One-way ANOVA with post-hoc Tukey's analysis).

Figure 2. Cluster diagram of transcripts differentially regulated by BMP-7 in cultured sympathetic neurons.

The 60 most significantly regulated transcripts (ANOVA, p<0.005) were analyzed by Partek Genomics Suite 6.5 to determine hierarchical clustering. Samples (columns) are clustered based on treatment condition whereas transcripts (rows) are clustered based on expression pattern. Relative levels of gene expression are depicted with a color scale in which red represents the highest level of up-regulated expression and blue represents the lowest level of down-regulated expression. Unsupervised clustering identified 4 major groups of genes, identified by the solid lines and numbers on the far right. Group 1 represents genes that are upregulated by 6 hr after BMP-7 treatment and remain upregulated at 24 hr after BMP-7 treatment; Group 2, genes upregulated only at 24 hr post-BMP-7 addition; Group 3, genes that are downregulated by BMP-7 at 24 hr; and Group 4, genes that are downregulated by 6 hr after BMP-7 treatment and remain downregulated at 24 hr after BMP-7 treatment. Additional information for the genes identified in Groups 1 and 2 is provided in Table S1; genes identified in Groups 3 and 4 are described in more detail in Table S2.

Figure 3. Overview of gene expression changes after addition of BMP-7 to sympathetic neurons.

Venn diagram showing concordance of significant changes in transcript levels between treatment comparisons. The numbers in each section refer to the number of annotated genes that were found to be significantly different between treatment groups (p<0.05, 1.2 fold change up or down). Specific genes corresponding to groups identified by the letters A through G are identified in Tables S3, S4, S5 available in on-line supporting information.

Figure 4. Functional analysis of genes differentially regulated during dendritogenesis in cultured sympathetic neurons.

Probe sets that were determined to be significantly regulated in sympathetic neurons exposed to BMP-7 for 6 hr (compared to controls) were analyzed using MetaCore software (GeneGo). The most significant Gene Ontology signaling and metabolic (top panel) and cellular and molecular (bottom panel) pathways are shown.

Figure 5. Informatic analysis of interactions between BMP-7-regulated genes identifies potential roles of Id transcriptional repressors.

Transcripts differentially regulated by BMP-7 at 6 hr (p<0.05, 1.2 fold change up or down; listed in Tables S3, S4, S5) were entered into the GenGO Analyze Networks (AN) algorithm with default settings. Shown is one of the top scored networks as identified by an enrichment z-score. Thick cyan lines indicate the fragments of canonical pathways. Up-regulated genes from the gene input list are marked with red circles. A significant network of pathways linked by annotated functional data included three of the Id genes, which are among the most robust transcriptional regulators identified in our analysis and which are found in the same cluster of genes activated at both 6 and 24 hr after BMP-7 treatment.

Figure 6. Validation of BMP-7 induction of expression for genes identified as up-regulated in microarray analysis.

To validate results of microarray analysis of gene expression, we analyzed gene expression in primary cultures of sympathetic neurons treated with or without BMP-7 for 24 hr. Total RNA collected from these cultures was submitted to Northern blot analysis using six independent probes corresponding to genes identified as upregulated by microarray analysis. (A) Representative Northern blots probed for Id1, Id3, Delta1, Ngfr (p75), Jagged1 and CXCR4. In all cases, a single band was detected at the expected size, and equal loading of mRNA was verified by probing with actin, which was considered a housekeeping gene. (B) Effect of transcriptional and translational inhibitors on BMP-7-stimulated gene expression in sympathetic neurons. Cultures treated with or without BMP-7 in the absence or presence of actinomycin-D or cycloheximide to inhibit mRNA or protein synthesis, respectively. Total RNA from these cultures was analyzed by Northern blotting for expression of Id1 and Id3. BMP-7 induced expression of both Id1 and Id3 was blocked by actinomycin-D, while cycloheximide enhanced the expression of both genes in response to BMP-7.